Method for preparing biologically active (pyro) glutamyl-histidyl-tryptophyl-seryl-tyrosyl-glycyl-leucyl-arginyl-prolyl-glycine amide

ABSTRACT

A method for preparing (pyro)glutamyl-histidyl-tryptophylseryltyrosyl-glycyl-leucyl-arginyl-prolyl -glycine amide which comprises reacting an oligopeptide fragment identified as (I) with an oligopeptide fragment identified as (II), each of which are prepared according to the synthesis described below, to form the heptapeptide, seryl-tyroxyl-glycyl-leucyl-arginyl-prolylglycine amide having some corresponding protective groups thereon, and coupling the resulting heptapeptide with protected tryptophane, histidine and (pyro)glutamic acid in turn at the nitrogen terminal of the each resulting peptide by stepwise elongation to result in a protected decapeptide, and then removing all the protective groups by treating the protected decapeptide with hydrogen fluoride: Fragment (I) Leucyl-(N-protected)arginyl-prolyl-glycineamide is prepared by coupling protected arginine with proline whose carboxyl group is protected, to produce arginyl-proline, coupling glycine amide with the resulted arginyl-proline to produce arginyl-prolylglycine amide and further coupling leucine with the Alpha nitrogen terminal of the resulting tripeptide and then removing only the protective group on the nitrogen terminal of the leucine portion, and Fragment (II) (N- and O-protected)seryl-(O-protected)tyrosyl-glycine is prepared by coupling a lower alkyl ester of glycine to result in a corresponding seryl-tyrosyl-glycine lower alkyl ester and hydrolyzing only the ester bond of the tripeptide, thus formed.

United States Patent 1191 I Sakakibara et al. I v

14 1 Dec.24, 1974 METHOD FOR PREPARING BIOLOGICALLY ACTIVE (PYRO) GLUTAMYL-HISTIDYL-TRYPTOPHYL- SERYL-TYROSYL-GLYCYL-LEUCYL- ARGINYL-PROLYL-GLYCINE AMIDE [76] Inventors: Shunpei Sakakibara, No. 23-3,

Fujishirodai Z-chome, Suita-shi,

I Osaka; Yuichi Kumahara, No.

22-10, Takarayama-machi, Toyonaka-shi, Osaka; Terutoshi Kimura, No. 14-3402, Ohara-m'achi, Ashiya-shi, Hyogo, all of Japan 22 Filed: Sept. 5, 1972 21 Appl. No.: 286,394 j 30 Foreign Application Priority Data Sept. 2, 197i Japan 46-67745 52 us. c1. 260/112.5, 424/177 [51] Int. Cl... C07cl03/52, C07g 7/00, A61k 27/00 [58] Field of Search 260/1 12.5

[56] References Cited UNITED STATES" PATENTS 3,780,014 12/1973 Flouret 260/1 12.5 3,784,535 l/l974 Flouret' 260/] 12.5 3,787,386 l/l974 Flouret et al. 260/ll2.5 3,790,554 7 2/1974 Flouret 260/ll2.5 3,803,117 4/1974 Flouret .J. 260/1 12.5

OTHER PUBLICATIONS Chang et al., J. Med. Chem, 15, 623-627 (1972). Matsuo et al., Biochem. Biophys. Res. Comm, 45, 822-827 (1971). Sievertsson et al., Biochem. Biophys. Res. Comm, 44, 1566-1571 (1971). Monahan et al., C. R. Acad. Sc. Paris, 273 D, 508 (1971), Baba et al., Biochem. Biophys. Res. Comm, 44, 459 (1971), July pub. date. Matsuo et al., Biochem. Biophys. Res. Comm, 43,

1334 1971 June pub. date.

Primary Examiner-Lewis Gotts Assistant Examiner-Reginald J. Suyat Attorney, Agent, or Firm-Sughrue, Rothwell, Mion, Zinn & Macpeak [57] ABSTRACT ,A method for preparing (pyro)glutamyl-histidyl- Leucyl-(N-protected)arginyl-p rolyl-glycineamide is prepared by coupling protected a'rginine with proline whose carboxyl group is protected, to produce arginyl-proline, coupling glycine amide with the resulted arginyl-proline to produce argiriyl-prolyl-glycine amide and further coupling leucine with the a-nitrogen terminal of the resulting tripeptide and then removing only the protective group on the nitrogen terminal of the leucine portion, and

; Fragment 11 (N- and O-protected)seryl-(O-protected)tyrosyl-glycine is prepared by coupling a lower alkyl ester of glycine to result in a corresponding seryl-tyrosyl-glycine lower alkyl ester and hydrolyzing only the ester bond of the tripeptide, thus formed.

1 Claim, 1 Drawing Figure PATENTED DEE24 I974 WAVE LENGTH (mp) METHOD FOR PREPARING BIOLOGICALLY ACTIVE (PYRO) GLUTAMYL-HlSTIDYL-TRYPTOPHYL-SERYL- TYROSY L-GLYCYLLEUCYL-ARGINYL-PROLYL- GLYCINE AMIDE BACKGROUND or THE INVENTION (hereinafter, LH) and the follicle-stimulating hormone Y (hereinafter, FSH). 1

With respect to the present recovery of (RH), it has been reported that R'H- is extracted from calves hypothalami and its ten amino acid sequence is as follows: (Pyro )Glu-His-TrpSer-Tyr-Gly-Leu-Arg-Pro-Gly- NH that is (pyro)g]utamyl-histidyl-tryptophyl-seryltyrosyl-glycyl-leucylarginyl-prolyl-glycine amide.

However, no synthetic production method nor physical properties of the present decapeptide has been established. Generally, the synthesis of a polypeptide is, though theoretically possible, very difficult and frequently unsuccessful even if component amino acids and the sequence thereof have been defined and shown. These difficulties stem from the unexpected physical properties of oligopeptide fragmentswhich are essential for a certain synthetic pathway planned or upon impossibilities in purification to remove byproducts resulting from the planned procedure.

Therefore, it is generally considered that thestepwise AOC- Scr-Tyr-Gly OEl;

MeOH-NaOII Bzl 1321 elongation method to couple amino acids one by one is almost suitable for synthesis of polypeptides. The stepwise elongation method. however, is far from acceptable for commercial production of polypeptides, except for synthesis for study.

While various methods to create peptide bonds have now been established but for the commercial production of such long chained polypeptides, it is necessary to lay out a synthetic pathway consisting of some fragmentation of long sequence or proper combination of such fragmentation procedures with stepwise elongation. In practice, there is thought to exist numerous combinatiions of fragments for production of such long chained polypeptides. In addition to the numerous ;combinations, most oligopeptide fragments would belong to a novel compound group, so the physical properties are, of course, unexpected. Moreover, there would be many problems to be solved for the establish- 'ment of an ideal and commercial synthetic pathway, for

example, what type of protective group is suitable for the fragments employed, or what type of coupling reactions could be used, etc.

As a result of our investigation of those problems for the commercial production of the presentdecapeptide, we the applicants propose a synthetic method as described below. The proposed synthetic pathway consists of an ideal combination of fragmentation, coupling reactions and protective groups suitable for the fragments.

, SUMMARY OF THE INVENTION Synthetic Pathway for Production of RH T05 I C FaCOOH AOC- Ser-Tyr-Gly OH Fragrnentfll) H- Leu-Arg- Pro-Gly -Nllz Fragmeritfl) V L I v lCFaCOOH Bzl Bzl l Ser-Tyr-Gly-Leu-Arg-Pro-tlly lNII2 I Bzl Tos lilzl Bzl W S CI Bzl CFJCOOH 'fos TIMI

C FaCOOH Z-V (Pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Glyl-NH:

H (P yro) Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NHz below:

Aoc: tertiary amyloxycarbonyl group Boc: tertiary butyloxycarbonyl group Tos: p-toluene sulfonyl group Bzl: benzyl group I Z: benzyloxycarbonyl group Su: residual moiety of succinic imide Np: p-nitro-phenyl group In the above schematic, the symbols used are defined WSCI: water soluble carbodiimides such as C Ha CHa

or the mineral acid salts thereof.

The initial letters of amino acids each represent a 50 group of NI-ICH(R)CO- in correspondence to c the symbol R.

(These symbols are used according to the above definition in the present specification.)

BRIEF DESCRIPTION-OF THE DRAWING The FIGURE 1 is a UV absorption spectrum of the RH prepared in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION The present synthetic method for production of RH has many characteristic advantages, as hereinafter described, in comparison with any other. fragmentations proposed to produce the same:

such as angiotencine I or II, bradykinin or callidin which have a similar length to RH. The purification employed in the present pathway is easily carried out through silica gel column chromatography using economically inexpensive solvents to yield an excellent amount of each intermediate product and also RI-I. Moreover, the present pathway contains 40 only a few purification processes.

b. The reactions employed for the formation of the peptide couplings do not cause racemization of the material amino acids used or fragments produced,

because the present fragmentation is so designed as to divide RH into two fragemnts I and II at the bond between the glycine portion and the leucin portion of RH. In addition, suitable acyloxy protective groups for the amino radical are employed so as to avoid formation of an oXazolidone ring in the coupling reactions.

fragments, particularly fragment I, provides intermediates suitable for handling, in manufacture (for instance, its hydrophilic-lipophilic properties) owing to the combination'of the order and the selected protective groups shown in the previous schematic. In addition to this, yields of byproducts, such as diketopiperazine (for example, another coupling order, Pro-Gly Arg-Pro-Gly, may cause diketopiperazine production and these intermediate fragments are too highly hydrophilic to be handled in the production of the fragments) do not occur. This proves that the present coupling order and employment of the protective groups are very advantageous to achieving the goals of this invention. d. In the present pathway, the activated ester method and WSCI-method are so properly employed in the order of couplings in the production of the which have some protective groups on the nitrogen or carbon terminal of the fragments.

With respect to the production of fragment II, the

protective group on the carbon terminal of glycine in.

fragment II is an ester moiety which is thought to bemost suitable. It is though that removal of the protective ester moiety might cause cleavages of the peptide bonds at the same time. However, a selective hydrolysis on the protective ester moiety actually proceeded successfully to yield fragment II without any side reaction.

The thus produced RH according to the present 'invention has been confirmed to be effective in the release of LH and FSH in accordance with biological animal tests, carried out by the'present inventors.

of 1.5 g of Pd-C, hydrogen gas was passed through the solution with stirring for 5 hours. Then Pd-C was re moved by filtration and the methanol was distilled off. The residue obtained was dissolved in 300 ml of AcOEt, and extracted with 5 percent NaHCO The extract was adjusted to a pH of 3 with 6N HCl under cool ing and re-extracted quickly with AcOEt. After washing with water, the extract was dried over anhydrous Na SO The Na SO was removed by filtration and the filtrate was concentrated to dryness. To the residue, there was added a large volume of Et O for precipita tion purposes. The precipitate was dried on P 0 under reduced pressure to-give 17 g (90 percent) of the desired product. were added. After adjusting to a pH of 7 with 0.4 ml of N-methylmorpholine, the reaction solution was stirred vigorously under cooling for 30 minaddition of a saturated Na SO solution, the resulting liquid was extracted with a large volume of AcOEt. The

AcOEt layer was washed with 0.5N HCl, H 0, 5%

A better understanding of the present invention will be gained from the following example, which is merely intended to be illustrative and not limitative of the present invention.

EXAMPLE (Preparation of Fragment I) (dimethylaminopropyl)-carbodiimide) was added to the solution under cooling at l0 --2C. The resulting mixture was adjusted to a pH of 7 byadding 0.53 ml of N-methylmorpholine, stirred'at 5C for minutes and at room temperature overnight. From the reaction mixture CH C1 was distilled off to give an oily product which was dissolved in CHCl The CHCl solution was washed with 0.5N HCl, H 0, 5% NaHCO and H 0 in turn, and then dried over anhydrous Na SO Na SO was removed by filtration and the filtrate was concentrated to give an oily product; The product was dissolved in. 150 ml of AcOEt with heating, and then the solution was placed in a refrigerator to precipitate crystals in the form of needles. The crystals were collected by filtration, washed with AcOEt and dried in a desiccator on P 0 under reduced pressure to give 24.0 g (73.4 percent) of the desired product. mp. 163.5-164.5C (a),, 33.4 (c 1.1; DMF).-

Analysis calculated for C ,l-l QN s c 59.13, H 6.s.N.11.12 found c 59.09, H 6.65. N 11 02 2. Synthesis of AoC-Arg(Tos)-Pro-OH In 200 ml of methanol, 22 g mmole) of Aoc- Arg(Tos)-Pro-OB21 were dissolved. After the addition NaHCO and H 0 in turn. In this process, the water layers were always saturated with Na SO The AcOEt layer was dried over MgSO MgSO was removed by a filtration, the filtrate was concentrated and the oily substance obtained was precipitated by the addition of ether. The precipitate was collected by filtration on a perature for 1 hour. The CF COOH was removed by distillation under reduced pressure, and the residue was dried in a desiccator on NaOH in vacuo to give an oily product (H-Arg-(Tos) Pro-Gly-NH .CF COOH). The oily product was dissolved in 2.5 ml of DMF, adjusted to a pH of 4 by addition of 2 ml of EtgN under icecooling. The solution was then adjusted to a pHof 7 by another addition of 1 ml of N-methylmorpholine, to which 1.7 g (5 mmole) of Boc-Leu-OSu and 1 ml of DMF were added. After stirring at room temperature for 17 hours, 1.5 ml of N,N- dimethylaminopropylamine were added to the solution and this was stirred at room temperature for 2 hours. The reaction mixture adding a large volume of water was extracted with CHCl The CHCl layer was washed with 0.5N HCl, H 0, 5 percent NaHCO and H 0, then dried over MgSO MgSO was removed by filtration and the filtrate was concentrated to dryness. To the residue there was added EtOH and AcOEt and the mixture was heated. After allowing to stand in a refrigerator, the precipitate crystallized and the crystals were ecollected by filtration, washed with sufficient AcOEt and dried in a desiccator on P 0 in vacuo to give 1.78 g (76.4 percent) of the desired compound. mp 1 50.5-152.5C (decomposedand became transparent at 182C). ((1) 7 33.0(c 1.0; DMF).

Analysis calculated for C;,,H ,O,,N S. /zH O C 52.89. H 7.31. N 15.92 found C 52.79, H 7.35, N 15.67

(Preparation of Fragment II) 1. Synthesis of Boc-Tyr(Bzl)-Gly-OEt 1n 5 ml of DMF there was added 500 mg of G1y- OEt.HCl. To this 0.5 ml of Et N was added with stirring under cooling. To the mixture there was added 1.45 g of Boc-Tyr(Bzl)-OSu. The mixture was stirred at room temperature for one day and diluted with CHCl After addition of 0.5 ml of dimethylaminopropylamine, the resultingmixture was stirred for 30 minutes. The reaction mixture was washed with N-HCl, H O,N Na CO and H 0, then the CHCl layer was dried over MgSO and CHCl was removed by distillation to yieldcrystals which were recrystallized from ethyl acetate-hexane to give 1.21 g (85.8 percent) of the desired compound. mp 131.5132.5C.

2. Synthesis of Aoc-Ser (Bzl)=Tyr(Bzl)-Gly-OEt 5 ml of CF COOH were added to 3.76 g of Boc- Tyr(Bzl)-Gly-OEt with stirring under cooling. After minutes this mixture was warmed up to room tempera ture and stirred for 40 minutes. CF COOH was distilled off in vacuo and the oily substance obtained was dried in a desiccator on NaOH. The substance was dissolved in 4 ml of DMF and the solution was adjusted to a pH of 7-8 by adding 264 ml of Et N with stirring under cooling. To this, 3.70 g of Aoc-Ser(Bzl)-OS4 were added and stirred at room temperature for 2 days. Then the reaction mixture was diluted with CHCl and washed with N HCl, H O, N Na CO and H 0. The CHCl layer was dried over MgSO and Cl-lCl was removed by distillation to give crystals which were recrystallized from ethyl acetate-hexane to give 4.54 g (84.1 percent) of the desired compound. mp 7l.574.5C.

Analysis calculated for C H O N C 66.74, H 7.00, N 6.48 found C 66.95, H 7.20, N 6.42

2. Synthesis of Aoc-Ser(Bzl)-Tyr(Bzl)-Gly-OH In 5 ml of MeOH, 2.02 g of Aoc-Ser(Bz l)-Tyr(Bzl)- Gly-OEt were dissolved. To the solution 3 ml of N NaOH were added dropwise with stirring and then it was rendered to react for 4 hours. After adjusting to a pH of 7 with N HCl, MeOl-l was removed. Then the solution was adjusted to a pH of 3 with another N HCl to precipitate crystals which were extracted with AcOEt. The AcOEt layer was washed with H 0 and dried over MgSO The AcOEt was removed by distillation to give crystals which were recrystallized from ethyl acetatehexane to give-3.54 g (80.4 percent) of the desired compound. mp 94.5-98.0C (00 -16. 1 (c 1.5; DMF).

Analysis calculated for C H o N C 65.89, H 6.67, N 6.78 found C 66.20. H 6.76, N 6.68

Amino acid analysis (6N HCl, 105C, 24 hours) Ser 0.95, Tyr 0.89, Gly 1.00.

(CONDENSATION OF FRAGMENTS 1 AND 11) Synthesis of Aoc-Ser(Bzl)-Tyr(Bzl)-Gly-Leu Arg(Tos)-Pro-Gly-NH To 694 mg of Boc-Leu-Arg(Tos)-Pro-Gly-NH there was added .3 ml of CF COOH with stirring under cooling. After stirring at room temperature for 40 minutes,

CF COOH was removed by distillation in vacuo. To the oily substance there wasadded ether to crystallize out the occurring white precipitate which was dried in a desiccator on NaOH. The precipitate was dissolved in a mixture of 20 ml of THF and 3 ml of CH CN and to the resulting solution there was added 0.17 mlof N- ethyl-N'(dimethylaminopropyl) carbodiimide with stirring under cooling at -15- 20C. This procedure was carried out at a pH of 7-8. To the mixture there was added 711 ml of Aoc-Ser(Bz1)-Gly-OH and the resu1ting mixture was stirred at room temperature for 30 minutes and allowed to stand overnight. Then the solvent was removed by distillation and the residue was dissolved in CHCl The CHCl; layer was washed with N HCl, H 0, 5 NaHCO and H 0 and then dried over MgSO Then the CHCI was distilled off to yield a powder of gel form. This was subjected to SiO column chromatography and eluted with a mixed solution of CHCl ):MeOH(5):AcOH(3). The obtained powder gave a single spot on TLC. The powder was reprecipitated from chloroform-ether to give 960 mg (78.7 percent) of the desired product. mp 1l8.5l23.0C (a) -29.1(c 1; DMF).

Analysis calculated for C H O N SH O C 59.29. H 6.97. N 12.67 found C 59.35. H 6.97. N 1220 Synthesis of Aoc-Trp-l-ll To 856 mg of Aoc-Ser(Bzl)-Tyr(Bzl)-Gly-Leu- Arg(Tos)-Pro-Gly-NH 5 ml of CF COOH were added with stirring under cooling. After 10 minutes, the mixture was warmed up to room temperature and stirred for 40 minutes. Excess CF COOH was removed by distillation and to the oily residue obtained, ether was added to crystallize out the occurring white precipitate. This was dried in a desiccator on NaOH and dissolved in 2 ml of DMF and the solution was adjusted to a pH of 8 by adding 0.2 ml of N-methyl-morpholine with stirring under cooling. After addition of 400 mg of Aoc- Trp-ONp, the solution was stirred at room temperature for 2 days and diluted with CHCl To the mixture, 0.5 ml of dimethylaminopropylamine were added and stirred for 45 minutes. The CHCI layer was washed with N-HCl, H O, N-Na CO and H 0, and dried over MgSO The CHCl was removed through distillation to yield a gel substance which was subjected to SiO column chromatography and eluted with a mixed solvent of CHCl (5):AcOEt(5): EtOl-l(2) to yield the desired product which gave a single spot on TLC. This was recrystallized from chloroform-ether to give 777 mg (78.7 percent) of the compound. mp 143.0-148.0C (a),,34.5 (c 1;DMF).

Synthesis of Boc-His(Tos)-Trp-l-Il To 549 mg of Aoc-Trp-Ser(Bzl)-Tyr(Bzl)-Gly-Leu- Arg(Tos)-Pro-Gly-NH 0.05 ml of mercaptoethanol (HSCH CH OH) and 5 ml of CF COOH were added. After 10 minutes, it was warmed up to room temperature and stirred for 40 minutes. Excess CF COOH was removed by distillation and ether was added to the oily residue to give a white precipitate. The precipitate was dried in a desiccator on NaOH and dissolved in a mixture of 2 ml of DMF. and 3 ml of CH CN. To the solution there was added 0.08 ml of N-ethyl-N-(dimethylaminopropyl)carbodiimide with stirring undercooling (-15--20C) then 226mg of Boc-His(Tos)-OH were added. The mixture was stirred at the same temperature for 1 hour, then warmed up to room temperature and allowed to stand overnight. The CH CN was removed through distillation in vacuo. CHCl was added and the CHCI layer was washed with N-l-lCl, H 0, 5 percent NaHCO and H 0 and then mg (59.3 percent) of the desired compound. mp

Analysis calculated for c n o u s ii o c 58.74, H 6.35, N found c 53.40, H 6.11 N

Synthesis of Z(Pyro)Glu-His(Tos)-Trp-l-Il To 252 mg of Boc-His(Tos)-Trp-Ser('Bzl)-Tyr(Bzl)- Gly-Leu-Arg(Tos)-Pro-Gly-NH 0.05 ml of mercatoethanol, mg of tryptophane and 3 ml of CF COOH were added with stirring under cooling. After 10 minutes, the resulting mixture was warmed up to room temperature and stirred for 40 minutes. Then an excess of CF COOl-l was removed by distillation. To the oily substance obtained, ether was added to crystallize out a white precipitate which was dried in adesiccator on NaOH and dissolved in 1 ml of DMF. The solution was adjusted to a pH of 7-8 by adding 0.15 ml of N- methylmorpholine with stirring under cooling. To this 86 mg of Z-( Pyro)Glu-ONp was added and the mixture was warmed up to room temperature,'and then stirred for 2 days. After addition of CHCl the reaction mixture was washed with N-HCl, H O, NNa CO and H 0. The CHCl layer was dried over MgSO, and the CHCL, was removedby distillation to give a substance in the form of a gel. The gel was subjected to SiO column chromatography, eluting with a mixed solvent of CHCl (95):MeOl-l(5):AcOH(3) to yield a compound which showed a single spot on TLC. This was reprecipitated from chloroform-ether to give 150 mg (54.6 percent) of the desired compound with a melting point of 133.5-138.5C (decomposed).

SYNTHESIS-OF RH I In the HF-reaction vessel, 100 mg of Z-(pyro)Glu- His( Tos)-Trp-Ser( Bzl )-Tyr( Bzl )-Gly-Leu-Arg( Tos)- Pro-Gly-NH were introduced. Then 51 mg of tryptophane,.21.6 mg of skatol, 0.6m] ofanisol, and 5 ml of 60 NaOH and then it was dissolved in cold water which was washed with ether. The water layer was passed through a column of an ion-exchange resin (Dowex lX2(AcO); trade name) which was eluted with H O. The water solution was freeze-dried to yield a powder which was treated with a chromatography of Sephadex G-15 (trade name) eluting 0.2 N AcOH. The main fractions were collected and freeze-dried to yield 25 mg of powder. When the powder was subjected to electrophoresis, a single spot was obtained, whose ninhydrin reaction was negative and Pauri reaction was positive. (a),,46.2 (c 0.3; H 0).

Analysis calculated for C l-l O N .3(CH COOH).4H O

C 51.07, H 6.68, N 16.60 found C 51.00, H 6.59, N 16.48

Amino acid analysis (6N HCl, 105C; 24 hours). Glu 1.11, His 0.90, Trp 0.94, Ser 0.97, Tyr 0.96, Gly 2.02, Leu 1.00, Arg 1.02, Pro 1.05. NOTE: The value of Trp was calculated from the absorbance of UV spectrum shown in FIG. 1.

Although the present invention has been adequately amide having some corresponding protective groups thereon, and coupling the resulting heptapeptide with protected tryptophane, histidine and (pyro)glutamic acid in turn at the nitrogen terminal of the each resulting peptide by-stepwise elongation to result in a prohydrogen fluoride:

Fragment (I): Leucyl-(N-protected)arginyl-prolyl-glycineamide is prepared by coupling protected arginine with proline whose carboxyl group is protected, to produce arginylproline, coupling glycine amide with the resulted arginyl-proline to produce arginyl-prolylglycine amide and further coupling leucine with the a-nitrogen terminal of the resulting tripeptide and then removing only the protective group on the nitrogen terminal of the leucine portion, and

Fragment (II):

glycine is prepared by coupling a lower alkyl ester T of glycine to result in a corresponding seryl-tyrosylglycine lower alkyl ester andlnydrolyzing only the ester bond of the tripeptide, thus formed. V

' l =l =i and O-protected)seryl-(D-protected)tyrosyl 

1. A METHOD FOR PREPARING BIOLOGICALLY ACTIVE (PYRO)GLUTAMYL-HISTIDYL-TRYPTOPHYI-SERYL-TYROSYL-GLYCYL-LEUCYL-ARGINYLPROLYL-GLYCINE AMIDE WHICH COMPRISES REACTING AN OLIGOPEPTIDE FRAGMENT IDENTIFIED AS (I) WITH AN OLIGOPEPTIDE FRAGMENT IDENTIFIED AS (II), EACH OF WHICH ARE PREPARED ACCORDING TO THE SYNTHESIS DESCRIBED BELOW, TO FORM THE HEPTAPEPTIDE, SERYLTRYROSYL-GLYCYL-LEUCYL-ARGINYL-PROLYL-GLYCINE AMIDE HAVING SOME CORRESPONDING PROTECTIVE GROUPS THEREON, AND COUPLING THE RESULTING HEPTAPEPTIDE WITH PROTECTED TRYPTOPHANE, HISTIDINE AND (PYRO)GLUTAMIC ACID IN TURN AT THE NITROGEN TERMINAL OF THE EACH RESULTING PEPTIDE BY STEPWISE ELONGATION TO RESULT IN A PROTECTED DECAPEPTIDE, AND THEN REMOVING ALL THE PROTECTIVE GROUPS BY TREATING THE PROTECTED DECAPEPTIDE WITH HYDROGEN FLUORIDE: FRAGMENT (I): LEUCYL-(N-PROTECTED)ARGINYL-PROLYL-GLYCINEAMIDE IS PREPARED BY COUPLING PROTECTED ARGININE WITH PROLINE WHOSE CARBOXYL GROUP IS PROTECTED, TO PRODUCE ARGINYLPROLINE, COUPLING GLYCINE AMIDE WITH THE RESULTED ARGINYL-PROLINE TO PRODUCE ARGINYL-PROLYL-GLYCINE AMIDE AND FURTHER COUPLING LEUCINE WITH THE A-NITROGEN TERMINAL OF THE RESULTING TRIPEPTIDE AND THEN REMOVING ONLY THE PROTECTIVE GROUP ON THE NITROGEN TERMINAL OF THE LEUCINE PORTION, AND FRAGMENT (II): (N- AND 0-PROTECTED)SERYL-(O-PROTECTED) TROSYL-GLYCINE IS PREPARED BY COUPLING A LOER ALKYL ESTER OF GLYCINE TO RESULT IN A CORRESPONDING SERYL-TYROSYL-GLYCINE LOWER ALKYL ESTER AND HYDROLYZING ONLY THE ESTER BOND OF THE TRIPEPTIDE, THUS FORMED. 